Equol-producing lactic acid bacteria-containing composition

ABSTRACT

An equol-producing lactic acid bacteria-containing composition comprises, as an essential component thereof, a lactic acid bacterial strain belonging to the genus Lactococcus having an ability to utilize at least one daidzein compound selected from the group consisting of daidzein glycosides, daidzein, and dihydrodaidzein to produce equol. Such a composition is effective for the prevention and alleviation of malaise inclusive of climacteric disturbance in middle-aged and elderly women for which no effective prophylactic method or alleviating means has heretofore been available.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation of U.S. application Ser. No. 10/562,687 filedDec. 28, 2005 (now U.S. Pat. No. 8,765,445), which is a National StageEntry of PCT International Application No. PCT/JP2004/009484 filed Jun.29, 2004, which claims benefit of Japanese Patent Application No.2003-187831 filed Jun. 30, 2003. The above-noted applications areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to equol-producing lactic acid bacterialstrain, a composition comprising said lactic acid bacterial strain, anda method of producing equol by utilizing said lactic acid bacterialstrain.

BACKGROUND OF THE INVENTION

It has heretofore been reported mostly in Europe and the United Statesof America that isoflavone (soy isoflavone) contained in soybeans hasprophylactic efficacies (antiestrogen effect) in breast cancer,carcinoma of the prostate, and other diseases and that it hasalleviating efficacies (estrogenic-like effect) in climacteric andpostmenopausal osteoporosis, hyperlipidemia, hypertension, etc. (H.Adlercreutz et al., (1992) Lancet, 339, 1233; H. Adlercreutz et al.,(1992) Lancet, 342, 1209-1210; D. D. Baird et al., (1995) J. Clin.Endocrinol. Metab., 80, 1685-1690; A. L. Murkies et al., (1995)Maturitas., 21, 198-195; and D. Agnusdei et al., (1995) Bone andMineral., 19 (Supple), S43-S48).

Recently, however, doubts have been cast on the clinical efficacy of soyisoflavone and, instead, it is reported that equol as the activemetabolite of soy isoflavone is a key factor in the expected efficaciesin clinical application. Thus, several reports are available arguingthat in breast cancer, carcinoma of the prostate, and climacteric andpostmenopausal osteoporosis, the efficacy of soy isoflavone is surpassedby that of equol, the metabolite of soy isoflavone (D. Ingram et al.,(1997) Lancet, 350, 990-994; A. M. Duncan et al., (2000) CancerEpidemiology, Biomarkers & Prevention, 9, 581-586; C. Atkinson et al.,(2002) J. Nutr., 32(3), 595S; H. Akaza et al., (2002) Jpn. J. Clin.Oncol., 32(8), 296-300; and S. Uchiyama et al., (2001) Ann. Nutr.Metab., 45, 113(abs)).

Moreover, many lectures were delivered on the subject of equol in the4th International Symposium on the Role of Soy in Preventing andTreating Chronic Disease (San Diego, USA, 2001), and in December 2002 acomprehensive review of studies on equol was also reported. Thus, it isgetting more or more accepted in academic circles that equol is the veryentity of efficacies of soy isoflavone (K. D. R. Settchell et al.,(2002) J. Nutr., 132, 3577-3584).

Furthermore, compared with soy isoflavone, equol is delivered to tissuessuch as the breast tissue and prostatic tissue with by far greaterefficiency and, from this fact, the physiological significance of equolis endorsed (J. Maubach et al., (2003) J. Chromatography B., 784,137-144; T. E. Hedlund et al., (2003) The Prostate, 154, 68-78).

Equol is produced by the intestinal flora and the involvement ofindividual difference in its production has been reported. It is alsoreported that equol producers among the Japanese account for about 50%(S. Uchiyama et al., (2001) Ann. Nutr. Metab., 45, 113 (abs)).Individuals who cannot produce equol are suspected to be lacking inequol-producing bacteria in their intestine. In such individuals, it issuspected that the expected antiestrogen and estrogenic-like effects maynot be expected even if processed soybean foods are ingested. In orderthat the expected effects may be expressed in such individuals, it seemsto be a reasonable course of action to have them ingest equol-producingbacteria or equol as such.

Based on the above idea, the inventors had conducted intensiveinvestigations and isolated from human stools novel 3 strains ofmicroorganisms and identified them: namely Bacterioides E-23-15 (FERMBP-6435), Streptococcus E-23-17 (FERM BP-6436), and Streptococcus A6G225(FERM BP-6437), as equol producing-bacteria suitable for the expressionof said antiestrogen and estrogenic-like effects, among other effects,and applied for a patent claiming inventions concerning theseequol-producing strains of microorganisms and utilization of themicroorganisms (WO99/07392).

DISCLOSURE OF INVENTION

The inventors conducted further studies and succeeded in the isolationand characterization of a lactic acid bacterial strain belonging to thegenus Lactococcus which are capable of utilizing daidzein glycoside,daidzein, or dihydrodaidzein to produce equol as a novel strain ofmicroorganism which is fundamentally different from the previouslyisolated and identified microorganisms. The present invention has beendeveloped on the basis of the above isolation and identification of thisnovel strain of lactic acid bacterium.

The present invention subsumes the following inventions summarized inparagraphs 1-13.

-   -   Item 1. An equol-producing lactic acid bacteria-containing        composition comprising, as an essential component thereof, a        lactic acid bacterial strain belonging to the genus Lactococcus        having an ability to utilize at least one daidzein compound        selected from the group consisting of daidzein glycosides,        daidzein, and dihydrodaidzein to produce equol.    -   Item 2. The composition according to Item 1, wherein said lactic        acid bacterial strain belonging to the genus Lactococcus is        Lactococcus garvieae.

Item 3. The composition according to Item 2, wherein said lactic acidbacterial strain belonging to Lactococcus is Lactococcus 20-92 depositedunder FERM BP-10036.

-   -   Item 4. The composition according to Item 1 further comprising        at least one member selected from the group consisting of        daidzein compounds and daidzein compound-containing ingredients.    -   Item 5. The composition according to Item 4, wherein the        daidzein compound-containing ingredient is soybean flour or soy        milk.    -   Item 6. The composition according to Item 4 which is in the form        of a beverage or a milk product.    -   Item 7. The composition according to Item 4 further comprising        equol.    -   Item 8. The composition according to Item 7 which is in the form        of a fermentation product of soy milk.    -   Item 9. A method of producing equil comprising the step of        letting a lactic acid bacterial strain belonging to the genus        Lactococcus having an ability to utilize a daidzein compound to        produce equol act on at least one member selected from the group        consisting of daidzein compounds and daidzein        compound-containing ingredients.    -   Item 10. The method according to Item 9, wherein said lactic        acid bacterial strain belonging to the genus Lactococcus is        Lactococcus garvieae.    -   Item 11. The method according to Item 10 wherein said lactic        acid bacterial strain belonging to the genus Lactococcus is        Lactococcus 20-92 deposited under FERM BP-10036.    -   Item 12. The method according to Item 9, wherein the daidzein        compound-containing ingredient is soybean flour or soy milk.    -   Item 13. A lactic acid bacterial strain belonging to the genus        Lactococcus as deposited under FERM BP-10036.

The equol-producing lactic acid bacteria-containing composition of theinvention described in detail bellow.

(1) The Lactic Acid Bacterial Strain Belonging to the Genus Lactococcus

The equol-producing lactic acid bacteria-containing composition of theinvention comprises, as an essential component thereof, lactic acidbacterial strain belonging to the genus Lactococcus having an ability(metabolic activity) to utilize at least one daidzein compound selectedfrom the group consisting of daidzein glycosides, daidzein, anddihydrodaidzein and thereby produce equol.

A specific example of said lactic acid bacterial strain is Lactococcus20-92 (FERM BP-10036) which the inventors isolated from human stools andidentified de novo.

The bacteriological characteristics of the lactic acid bacterial strainare described in detail bellow.

I. State of Growth on the Medium

This strain shows good or normal growth on EG (Eggerth-Gagnon) agar, BL(Blood Liver) agar, and GAM (Gifu Anaerobic Medium) when culturedanaerobically in an anaerobic jar with steel wool at 37° C. for 48 hoursor cultured aerobically at 37° C. for 48 hours. The colonial morphologyis raised in a circular or convex manner, with both the surface andperipheral edge being smooth, and assumes a gray-white color on EG agarand a tan-brown color on BL agar. Morphologically, it is a Gram-positivediplococcus. This strain is not sporogenic.

II. Biochemical Characteristics

-   -   (1) Optimum temperature for growth: 37° C.    -   (2) Optimum pH for growth: 7.0    -   (3) Liquefaction of gelatin: −    -   (4) Production of acetoin from pyruvic acid: +    -   (5) Hydrolysis of hippuric acid: −    -   (6) Hydrolysis of esculin: +    -   (7) Pyrrolidonyl arylamidase: +    -   (8) α-Galactosidase: −    -   (9) β-Galactosidase: −    -   (10) β-Glucronidase: −    -   (11) Alkaline phosphatase: −    -   (12) Leucine arylamidase: +    -   (13) Arginine dihydrase: +    -   (14) Assimilation of carbon sources        -   D-Ribose +        -   L-Arabinose −        -   D-Mannitol +        -   D-Sorbitol −        -   Lactose −        -   D-Trehalose +        -   Inulin −        -   D-Raffinose −        -   Starch +        -   Glycogen −            (15) Organic Acid Composition after Utilization of Peptone            or Glucose

Using PYF (peptone-yeast extract Fields) medium (peptone content: ca 5%)used as sugar utilization medium and PYF medium supplemented withglucose at a final concentration of 0.5%, the strain of the inventionwas cultured aerobically at 37° C. for 72 hours and the organic acids inthe cultures were assayed by HPLC. The results (unit: mM) are presentedbelow in Table 1

TABLE 1 Organic acid Peptone Glucose Maleic acid nd nd Succinic acid0.00 0.01 Lactic acid 3.33 27.35  Formic acid 1.13 0.88 Acetic acid 3.320.57 Pyroglutamic acid 0.12 0.25 Propionic acid nd nd i-Butyric acid ndnd n-Butyric acid nd nd i-Valeric acid nd nd n-Valeric acid nd nd nd =not detected

From the above cultural and biochemical characteristics, the strain ofthe invention is classified into Lactococcus garvieae which is agram-positive coccus but differs from its type strain (Schleifer, K. H.,Kraus, J., Dvorak, C., Kilpper-Balz, R., Collins, M. D. and Fischer, W.Transfer of Streptococcus lactis and related streptococci to the genusLactococcus gen. nov. Syst. Appl. Microbiol., 6, 183-195, 1985;ATCC43921 (JCM10343) and ATCC49156 (JCM8735)) in the utilization ofstarch.

Therefore, the inventors named this strain Lactococcus 20-92 anddeposited it with the National Institute of Advanced Industrial Scienceand Technology International Patent Organism Depositary, AIST TsukubaCentral 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken 305-8566, Japanas of Jan. 23, 2003, under the accession number of FERM P-19189. Thismicroorganism is now placed under Budapest Treaty deposit, and theaccession number is FERM BP-10036.

This strain was found to assimilate glucose and, then, elaborate lacticacid (L-lactic acid), verifying that it is a member of homofermentativelactic acid bacteria.

Furthermore, sequencing of the 16SrRNA of this strain revealed 99.189%homology with the type strain Lactococcus garvieae (JCM10343) and99.375% homology with Enterococcus seriolicida, JCM8735).

Incidentally, since Enterococcus seriolicida referred to above was akinto Lactococcus garvieae in DNA-DNA homology analysis, it wasreclassified into Lactococcus garvieae in 1996. Therefore, Lactococcusgarvieae has two type strains (JCM8753 strain and 10343 strain) whichare dissimilar in origin. Enterococcus seriolicida (JCM8735) was derivedfrom the kidney of infected yellowtail and the intrinsic Lactococcusgarvieae (JCM10343) was derived from bovine mastitis.

To explore the relative homology of Lactococcus 20-92 with the above twotype strains, a phenotypic comparison was made. The results arepresented below in Table 2.

TABLE 2 Strain of Characteristics invention JCM10343 JCM8735 Deamination± ± ± (Arg → NH₃) Temperature dependence of growth 10° C. + + + 15°C. + + + 30° C. + + + 37° C. + + + 40° C. + + − 45° C. − − − pHdependence of growth pH4.5 ± ± ± pH7.5 + + + pH9.6 + + + Salt tolerance(NaCl) 6.5 + + + Peptidoglycan Lys-Ala-Gly Lys-Ala-Gly Lys-Ala-GlyQuinone type MK-8,9 MK-8,9 No quinone MK denotes menaquinone

It will be apparent from Table 2 that the present strain Lactococcus20-92 was in agreement with the type strain (JCM10343) of Lactococcusgarvieae in phenotype but was different from the type strain (JCM8735)of Enterococcus seriolicida in growth behavior at 40° C. and in theproduction or non-production of quinone. Accordingly the present strainwas judged to be akin to Lactococcus garvieae (JCM10343).

The present strain Lactococcus 20-92 can be classed among Milk-culturedLactococci in the GRAS [acronym of Generally Recognized As Safe list byFDA (Food and Drug Administration, U.S.A.)] and its safety as a food isconsidered to be high.

With regard to Lactococcus garvieae, there is not a report eversuggesting its pathogenicity to human beings and there is no productionof virulent substances such as toxins, either, so that this bacterialspecies is generally acknowledged to be a species of high safety.

Moreover, Lactococcus garvieae has so far been detected in mozzarellacheese, raw milk, processed meat products under low-temperature storage,plaa-som (a fermented fish product widely consumed in Thailand), andToma Piemontese cheese which is an artisanal protected denominationorigin (PDO) Italian cheese, i.e. traditional cheese in Italy, amongothers, and reportedly this microorganism is detected at a highincidence of the order of 10⁵ cells/gram from plaa-som and 10⁸cells/gram from Toma Piemontese cheese, but this food has a long historyof eating assuring the safety of this organism. (P-M. Christine et al.,(2002) Int. J. Food Microbiol., 73, 61-70; M. G. Fortina, et al.,(2003), Food Microbiol., 20, 379-404).

On the other hand, Enterococcus seriolicida is reportedly pathogenic tocultured fishes such as yellowtail. Since Lactococcus 20-92, which is,thus, a strain of Lactococcus garvieae, is considered to bephylogenetically related to Enterococcus seriolicida, there was anapprehension of its pathogenic potential to cultured fishes. However,the studies by the inventors comparing the electronmicrograph ofLactococcus 20-92 with that of the pathogenic counterpart (Enterococcusseriolicida KG) revealed that unlike said pathogenic strain, the strainof the invention has no capsule on the cell surface. Therefore, thepresent strain is considered to have no pathogenicity, nor does itpresent with an ecological contamination problem. This conclusion isalso corroborated by the description in the following literature. Thus,Yoshida et al. argue about the pathogenicity of microbial cells tocultured fishes that a capsule present on the cell surface inhibitsphagocytosis by macrophages, with the result that the particularbacteria are not killed but septicemia is induced systemically incultured fishes infected with the bacteria (T. Yoshida, et al., (1996)Dis. Aquat. Org., 25, 81-86).

Furthermore, the present strain Lactococcus 20-92 retains the desiredequol-producing ability (activity) even in the case of directfermentation in milk and has the characteristic that for the maintenanceof this equol-producing ability, no special culture medium is required.Thus, by carrying out a fermentation in soy milk as used alone, thestrain utilizes daidzein compounds in the soy milk to elaborate equol.

Heretofore, there is no report available on lactic acid bacteria of thegenus Lactococcus which ever have such an equol-producing ability.Therefore, the present invention further provides a novel strain oflactic acid bacterium having such an equol-producing ability.

(2) Daidzein Compounds and Daidzein Compound-containing Ingredients

Daidzein compounds, which are utilized by the present strain Lactococcus20-92, include a daidzein glycoside, daidzein, and dihydrodaidzein. Aspecific example of said daidzein glycoside is daidzin. Daidzin is anisoflavone glycoside having daidzein as the aglycone (daidzeinglycoside). Referring to daidzin, it is utilized by said strain ofmicroorganism to liberate daidzein which is further utilized by thestrain to give dihydrodaidzein, from which equol is finally produced.

In the present invention, said daidzein compound is used as thesubstrate. The substrate includes not only daidzein compounds but alsovarious materials or ingredients containing the same. As arepresentative example of said material or ingredient containing saiddaidzein compounds (referred to as daidzein compound-containingingredient), soy isoflavone can be mentioned. Soy isoflavone is alreadyavailable from commercial sources and, in the present invention, suchcommercial products, for example “Fujiflavone P10” (registeredtrademark) from Fujicco Co., Ltd., can be used. Moreover, said daidzeincompound-containing ingredient includes not only soy isoflavone but alsoplant tissues as such, e.g. kudzu(=Pueraria thurbergiana Benth) and rootof kudzu (arrowroot), red clove, alfalfa, etc., and isoflavonederivatives originating therefrom.

Further examples of said daidzein compound-containing ingredientincludes not only the above-mentioned food materials such as soybean,kudzu, root of kudzu, red clove, alfalfa, etc. but also processedproducts thereof, such as soybean meal (soybean flour), boiled soybeans,tofu (soybean curd), fried bean curd, soy milk, soybean hypocotylextract, etc., fermentation products thereof, such as natto (fermentedsoybeans), soy sauce, miso, tempeh, and fermented soy beverages. Thesematerials invariably contain daidzein compounds. Moreover, these notonly contain daidzein compounds but also estrogenic isoflavones, such asgenistein and its glycosides (genistin etc.); glycitein and itsglycosides (glycitin etc.); biochanin A and formononetin which arepartially methylated daidzein and genistin precursors and can be usedwith advantage in the present invention.

(3) Composition of the Invention

(3-1) An Equol-Producing Lactic Acid Bacteria-containing Composition

The equol-producing lactic acid bacteria-containing composition of theinvention comprises, as an essential component thereof, acetic acidbacterial strain belonging to the genus Lactococcus having an ability toact on the substrate daidzein compound or daidzein-containing ingredientto produce equol, with the above-mentioned Lactococcus 20-92 being arepresentative example. The lactic acid bacterial strain for use as saidessential component usually are viable bacterial strain but are notlimited to these but may be any of its cultures, crude or purifiedpreparations of such cultures, which contains isolated cells, andlyophilizates thereof.

The cultures of said bacterial strain can be obtained typically by theprocedure comprising culturing the strain in a medium suited for itsgrowth, for example MRS medium, at 37° C. for about 48 hours. Followingthe cultivation, the cells can be recovered by, for example,centrifuging the culture at 3,000 rpm (4° C.) for 10 minutes. These canbe purified in the conventional manner. Moreover, these cells can belyophilized. The resulting lyophilizates can also be utilized as theactive component of the composition of the invention.

All that is necessary for the composition of the invention is that itcontains the bacteria (cells or equivalent) as said active componentbut, if desired, the composition may be supplemented with nutrientssuited for the maintenance (or growth) of the microorganism as saidactive component. The nutrients mentioned above may for example be thenutrient media for culture of the respective microorganisms, such asBHI, EG, BL and GAM, as mentioned hereinbefore.

Examples of the other nutrients include various oligosaccharides, suchas lactooligosaccharide, soy oligosaccharide, lactulose, lactitol,fructooligosaccharide, and galactooligosaccharide. The amount of sucholigosaccharides is not particularly restricted but is preferablyselected from a range such that the final concentration thereof in thecomposition of the invention will be about 1-3 weight %.

The above composition of the invention, when taken orally, expresses thedesired equol-producing activity in the recipient's body. Generally theJapanese have the habit to eat daidzein compound-containing foods,typically the above-mentioned food materials or ingredients, e.g.soybeans, secondary products thereof, and fermentation products thereofand, therefore, the intake of the composition of the invention resultsin the production of equol in vivo.

Furthermore, where necessary, the composition of the invention may besupplemented with suitable amounts of various vitamins, trace metalelements, and so forth. Examples of said vitamins include vitamin B,vitamin D, vitamin C, vitamin E, and vitamin K [particularly MK-7(menaquinone-7) derived from Bacillus natto]. Examples of said tracemetal elements are zinc, selenium, iron, manganese, etc.

The quantity of the microorganism to be formulated in the composition ofthe invention can be judiciously selected according to the kind ofbacterial strain used. Taking Lactococcus 20-92 as an example, thenumber of organisms (viable cell count) is preferably adjusted to about10⁸˜10⁹ cells/100 g composition. The viable cell count is determined asfollows. A sample dilution is coated onto an agar medium for bacterialculture and cultured aerobically at 37° C. and the colonies formed arecounted. The quantity of the microorganism described above can bejudiciously adjusted according to the form of the composition to beprepared using the above-mentioned quantity as a reference.

(3-2) The Daidzein Compound-containing Composition of the Invention

The composition of the invention may further contain, if necessary, atleast one member selected from the group consisting of theaforementioned diadzein compounds and daidzein compound-containingingredients. Among various kinds of daidzein compound and daidzeincompound-containing ingredient, soybean hypocotyls and food ingredientsprepared starting with the hypocotyls, are particularly preferred, and,among these, water-soluble or emulsified food ingredients are still morepreferred. Among other preferred examples of said daidzeincompound-containing ingredient are soybean flour and soy milk.

Due to the substrate contained in the formulation, a person notaccustomed to eating soybeans and the like food ingredients can take thecomposition orally, and the ingested microorganism utilizes theformulated substrate to produce the objective equol in the body.

The amount of said daidzein compound and/or daidzein compound-containingingredient in the composition is not particularly restricted but mayreasonably be about 10-25 mg which is equivalent to the usual dailyintake by the average Japanese.

(3-3) Equol-containing Composition of the Invention

The composition of the invention may further contain equol.

Generally, one's appetite for a food is whetted when the food materialis caused to undergo lactic acid fermentation, for instance. Moreover,Lactococcus 20-92, which is a representative example of microorganismfor use in the composition of the invention, has a very highequol-producing ability (activity). The present invention furtherprovides an equol-containing composition, such as fermented soy milk,which is prepared by permitting said strain of microorganism to act on adaidzein compound-containing ingredient such as soy milk, and therebylet it utilize the daidzein compound in the soy milk to elaborate equol.

As the substrate for use in this aspect of the invention, theabove-mentioned various kinds of daidzein compound and daidzeincompound-containing ingredient can be employed. Among these, solutionsor emulsions prepared from soy milk, soy flour or the like arepreferred.

A preferred specific example of the equol-containing composition of theinvention is the fermentation product obtained by a process whichcomprises adding soy isoflavone or a food material containing it to asuitable medium and culturing the microorganism of the invention,preferably Lactococcus 20-92, therein to cause fermentation. Moreparticularly, such fermentation can be effected by the procedure whichcomprises adding a predetermined amount of the microorganism of theinvention to a mixture of a sterilized substrate solution and a nutrientmedium favorable for growth of the microorganism, such as BHI, EG, BL orGAM, or to cow's milk, soy milk, or a vegetable juice which can be usedas food, and carrying out an anaerobic or aerobic fermentation reactionat 37° C. under stationary conditions for about 48-96 hours [wherenecessary, a pH control agent and a reducing agent (e.g. yeast extract,vitamin K₁, or the like) may be added]. In the above procedure, theamount of the substrate may be about 0.01-0.5 mg/ml, and the inoculumsize of the microorganism can be selected from the range of about 1 toabout 5%.

In this manner, the equol-containing composition of the invention can beproduced. This composition can be applied with advantage in theabove-described form of a fermentation product as a food or apharmaceutical product. Moreover, the produced equol can be isolated andpurified from the culture broth or fermentation product in the per seknown manner, optionally formulated with suitable amounts of other foodingredients or the like, and processed into suitable food forms orpharmaceutical product forms.

The isolation and purification referred to above can be achieved by, forexample, adsorbing the fermentation product on an ion exchange resin(DIAION HP20, product of Mitsubishi Kasei Corporation), eluting it withmethanol, and concentrating the eluate to dryness.

The amount of equol in the composition of the invention is selectedaccording to the form of food or pharmaceutical product to be producedand is not particularly restricted. Preferably, however, the amountshould generally be such that about 2-5 mg of equol will be contained ineach 100 g of the total composition.

The presence of equol in the product composition of the invention can beconfirmed by the method described hereinafter in Test Example 1.

The equol-containing composition of the invention ranks high on thesafety scale because the active ingredient equol is anaturally-occurring substance. Moreover, because it is prepared by usingthe lactic acid bacterial strain, the risk of contamination withchemicals originating from the production line is low. As additionaladvantages, high yield and low cost as well as savory taste and flavoras food can be mentioned.

(3-4) Forms of Food

The equol-producing lactic acid bacteria-containing composition of theinvention is generally processed into food forms comprising theparticular lactic acid bacterial strain as an essential component incombination with a suitable edible carrier.

Specific food forms of the composition of the invention include thebeverage form, milk product form other than said beverage form(inclusive of fermented milk form), solid food form, cell-containingmicroencapsulated form, and so forth. The composition of the inventionin the beverage form includes lactic acid bacteria beverages and lacticacid bacteria-containing beverages.

The terms “fermented milk” and “lactic acid bacteria beverage” as usedherein are in conformity with the definitions in Article 2-37 “FermentedMilk” and Article 2-38 “Lactic Acid Bacteria Beverage” of the“Regulations relating to the Ingredients etc. of Milks and MilkProducts” of the former Ministry of Health and Welfare. Thus, “fermentedmilk” means a pasty or liquid preparation resulting from thefermentation of a milk or a milk (dairy) product with lactic acidbacteria or yeasts. Therefore, the “fermented milk” includes not onlyproducts of beverage form but also products of yogurt form. The “lacticacid bacteria beverage” means a beverage prepared by using a pasty orliquid product resulting from the fermentation of a milk or a milkproduct with a lactic acid-fermenting bacterium or yeast as a chiefmaterial and diluting it with water.

The lactic acid bacteria-containing beverage includes fermentedvegetable drinks, fermented fruit drinks, and fermented soy milk drinksetc. Examples of items in the form of milk products other than thebeverage form includes products of the curd form such as yogurt. Thesolid food form includes granules, powders (inclusive of e.g.freeze-dried powders of fermented milk), tablets, effervescent tablets,gums, gumdrops, and puddings etc.

Processing into these forms can be carried out in the conventionalmanner. Moreover, the carrier for use in the processing into such formsmay be any edible carrier. Particularly preferred carriers are thosehaving good mouth-feel and taste-improving effects. Examples of suchcarriers having good mouth-feel and taste-improving effects includeartificial sweeteners, sorbitol, xylitol, and so on. Other preferredcarriers are, for example, masking agents such as trehalose (product ofHayashibara), cyclodextrin, Benekote BMI (product of Kao Corporation),etc.

The lactic acid bacteria-containing beverage as a preferred specificfood form is described in detail below. Processing into such a beveragecan be carried out by the procedure which comprises culturing themicroorganism in a suitable fermentation material containing nutrientsfor the microorganism, such as fluids derived from vegetables or fruits,soy-milk (emulsified soy), etc. to thereby cause fermentation of saidmaterial. The vegetables and fruits for use as the fermentation materialinclude cuttings, crushings, grindings, squeezed-out juices,enzyme-treated products, and dilutions or concentrates thereof. Thevegetables include pumpkins, carrots, tomatoes, sweet peppers, celery,spinach, pigmented sweet potatoes, corn, beats, kale, parsley, cabbages,and broccoli etc. The fruits include apples, peaches, bananas,strawberries, grapes, water melons, oranges, and mandarin oranges etc.

The cuttings, crushings, and grindings of vegetables and fruits can beobtained by, for example, the procedure which comprises washing thevegetable or fruit, subjecting it to a blanching treatment, e.g. placingin hot water, where necessary, and cutting, pulverizing or milling it bymeans of a crusher, mixer, food processor, pulper finisher, Mycolloider,or the like. Juices can be prepared by using a filter press,juicer-mixer, or the like. Juices can also be prepared by filtering saidgrindings (millings) through a filter cloth or the like. Theenzyme-treated products can be prepared by permitting cellulase,pectinase, protopectinase or the like to act upon said cuttings,crushings, grindings, or juices. The dilutions include 1 to 50-foldaqueous dilutions. The concentrates include those concentrated 1 to100-fold by such means as freeze concentration, concentration underreduced pressure, etc.

Soy milk which is another specific example of the fermentation substratematerial can be prepared from soybean materials in the routine manner.The soy milk includes a homogenate prepared by immersing skinnedsoybeans in water, wet-pulverizing these soybeans with a suitable millor the like, and homogenizing the pulverizate in the routine manner anda solution of water-soluble soy protein in water etc.

The fermentation using a microorganism can be carried out by inoculatingsaid fermentation substrate material with the microorganism of theinvention and incubating the inoculated material under stationaryconditions. The medium may optionally be supplemented withfermentation-promoting substances insuring good growth of themicroorganism used, for example various carbon sources such as glucose,starch, sucrose, lactose, dextrin, sorbitol, fructose, etc., nitrogensources such as yeast extract, peptone, etc., vitamins, and minerals.

The inoculum size of the microorganism should be generally equivalent toa viable cell count of not less than about 1×10⁶ cells, preferably about1×10⁷ cells per cubic centimeter of the fermentation substrate fluid. Asregards cultural conditions, the fermentation temperature is generallyselected from the range of about 20-40° C., preferably about 25-37° C.,more preferably 37° C. and the fermentation time is selected from therange of about 8-24 hours.

For stable fermentation, it is recommendable to prepare a starter inadvance and inoculate the fermentation substrate material with thestarter for fermentation. The representative starter may for example beculture obtained by inoculating the present strain of microorganism ofthe invention into said fermentation substrate material subjected tousual sterilization at 90-121° C. for 5-20 minutes beforehand, yeastextract-supplemented 10% skim milk powder, or the like and cultivatingthe microorganism under the same conditions as above. The starter thusprepared usually contains about 10⁷-10⁹ cells of the microorganism ofthe invention per gram of the culture.

The lactic acid fermentation product obtained in the above manner may attimes be a curd form (a yogurt-like or pudding-like form) and such aproduct can be directly taken as a food. The lactic acid fermentationproduct in said curd form can be further homogenized to prepare thedesired beverage form (for example, a fermented soy milk beverage). Thishomogenization can be carried out using an ordinary homogenizer. Moreparticularly, it can be carried out using Gaulin's high-pressurehomogenizer [LAB 40] at about 200-1000 kgf/cm², preferably about 300-800kfg/cm², or a Sanwa Machine Industry Co.'s homogenizer (article number:HA x 4571, H20-A2 etc.) at not less than 150 kg/cm². By thishomogenization, a beverage product, particularly a fermented soy milkbeverage, which has an excellent palatability, particularly a smoothmouth-feel, can be obtained. In carrying out said homogenization, it ispermissible, where necessary, to make appropriate diluting, add anorganic acid for pH adjustment, and/or add various additives which areusually employed in the manufacture of beverages, such as sugars, fruitjuices, viscosity builders, surfactants, and flavors, in suitableamounts. As a specific preferred example of each kind of additivementioned above and its addition level (% by weight based on the weightof the curd-form fermentation product) are: glucose 8% (% by weight, thesame applies hereinafter), sugar 8%, dextrin 8%, citric acid 0.1%,glycerol fatty acid ester 0.2%, and flavor 0.1%.

Thus obtained lactic acid bacteria beverage of the invention such as afermented soy milk beverage can be aseptically dispensed into suitablecontainers in the conventional manner to provide the end-product. Thisproduct has a good palatability allowing smooth swallowing and a goodflavor.

The dosage (intake amount) of the above product can be judiciouslyselected according to the age, sex, body weight, and severity of illnessof the recipient, among other variables, and is not particularlyrestricted. Generally, 100-300 mL of a beverage product with a viablecell count of 10⁸-10⁹ cells/mL can be ingested per day.

A further specific example of the composition of the invention in thefood form is the effervescent tablet form. This form can be prepared byformulating 10-35% (% by weight; the same applies below) of sodiumcarbonate and/or sodium hydrogencarbonate and 20-70% of a neutralizer,as effervescent ingredients, with 0.01-50% of the bacteria (lyophilizedcells) of the invention. The neutralizer to be used in this manner is anacidic compound capable of neutralizing said sodium carbonate and/orsodium hydrogencarbonate to generate carbon dioxide gas. Representativeexamples of said neutralizer are organic acids such as L-tartaric acid,citric acid, fumaric acid and ascorbic acid.

The amount of said effervescent ingredients in the effervescent productof the invention is such that when this product of the invention isdissolved in water, the solution shows acidity, particularly an acidityof pH about 3.5-4.6. More particularly, the amount can be selected fromthe range of 10-35% sodium carbonate and/or sodium hydrogencarbonate and20-70% neutralizer. Particularly, the amount of sodium carbonate isselected from the range of 11-31%, preferably 22-26%; sodiumhydrogencarbonate from the range of 10-35%, preferably 20-30%. Amongthese alternative choices, it is most preferable to use sodiumhydrogencarbonate alone within the range of 20-25%. The amount of theneutralizer is selected from the range of 20-70%, preferably 30-40%. Inparticular, it is most preferable to use L-tartaric acid within therange of 20-25% and ascorbic acid within the range of 8-15%.

The effervescent product contains the microorganisms of the inventionand the effervescent ingredients as essential components and mayoptionally be contained suitable amounts of various known additives suchas the excipient, binder, disintegrator, lubricant, viscosity builder,surfactant, osmolarity modulating agent, electrolyte, sweetener, flavor,colorant, pH control agent, and so forth. Examples of the additives arestarches such as wheat starch, potato starch, corn starch, dextrin,etc.; saccharides such as sucrose, glucose, fructose, maltose, xylose,lactose, etc.; sugar alcohols such as sorbitol, mannitol, maltitol,xylitol, etc.; sugar rearrangement glycosides such as coupling sugar,palatinose, etc.; excipients such as calcium phosphate, calcium sulfate,etc.; binders/thickeners such as starches, saccharides, gelatin, gumArabic, dextrin, methylcellulose, polyvinylpyrrolidone, polyvinylalcohol, hydroxypropylcellulose, gum xanthan, pectin, gum tragacanth,casein, alginic acid, etc.; lubricants such as leucine, isoleucine,L-valine, sugar esters, hydrogenated oils, stearic acid, magnesiumstearate, talc, macrogols, etc.; disintegrators such as crystallinecellulose (Avicel, Asahi Chemical Industry Co., Ltd.),carboxymethylcellulose (CMC), carboxymethylcellulose sodium (CMC-Na),carboxymethylcellulose calcium (CMC-Ca), etc.; surfactants such aspolyoxyethylene sorbitan fatty acid ester (polysorbate), lecithin, etc.;dipeptides such as aspartame, alitame, etc.; and sweeteners such asstevia, saccharin, and so forth. These can be judiciously selected andused in suitable amounts taking into consideration the relationship ofeach to the essential components, the proportion of the preparation, andmethod of production of the preparation, among other factors.

Furthermore, in the effervescent preparation of the invention, vitamins,particularly cyanocobalamine and ascorbic acid (vitamin C), can beformulated in suitable amounts. The amount is not particularlyrestricted but usually vitamin C, for instance, may be added up to 30%at the maximum, preferably within the range of about 5-25%.

The method of producing the effervescent preparation of the inventioncan be fundamentally similar to the conventional method for productionof effervescent tables of this kind. Thus, the preparation of theinvention in the effervescent tablet form can be prepared by weighingout predetermined amounts of the respective ingredients, mixing them,and processing the whole by the direct powder compression method or thedry or wet granulation-compression method, for instance.

The preparation of the invention, thus obtained, can be converted to abeverage form suitable for oral administration by mere placing in waterand be administered orally.

The dosage (intake amount) thereof can be judiciously establishedaccording to the age, sex, body weight, severity of illness of therecipient, among other variables, and is not particularly restricted butgenerally 1-2 tablets of the effervescent tablet form of the inventionweighing about 1.5-6.0 g per tablet can be dissolved in 100-300 mL ofwater and caused to be ingested per dose.

The particularly preferred blending proportions of the substratedaidzein compound or daidzein compound-containing ingredient, theparticular lactic acid bacterial strain, and optionally formulated otheringredients in the composition of the invention, per 100 g of thecomposition, are: the daidzein compound or daidzein compound-containingingredient within the range of about 10-50 mg calculated as daidzein,the number of the lactic acid bacterial strain within the range of10⁹-10¹⁰ cells (viable cell count), and oligosaccharides and otherswithin the range of about 1-5 g.

Since the equol-producing lactic acid bacteria-containing composition ofthe invention is designed to contain a microorganism (primarily livebacteria) as mentioned hereinbefore, such conditions as the applicationof heat and pressure are not recommendable in the processing of thecomposition into end products. Therefore, in processing the compositionof the invention into such product forms as bars, granules, powders, andtablets, it is preferable to directly formulate the microorganism in theform of lyophilized cells or use lyophilized cells treated with asuitable coating agent.

However, the equol-producing lactic acid bacteria-containing compositionof the invention need not essentially contain viable bacteria. When saidcomposition comprising viable bacteria and said daidzein compound or thelike which said bacteria may utilize contains bacteria-produced equol,it may be subjected to a routine heat sterilization to kill thebacteria. Such a heat sterilization given to the composition inhibitstaste and flavor deteriorations caused by excessive fermentation of theviable bacteria formulated in the composition during storage ordistribution on the market.

(3-5) Pharmaceutical Product Forms

The equol-producing lactic acid bacteria-containing composition of theinvention can be processed into pharmaceutical preparations generallycontaining said defined lactic acid bacterial strain as an essentialcomponent together with a suitable pharmaceutically acceptable carrier.

The carrier includes various diluents and excipients, such as fillers,volume builders, binders, humectants, disintegrators, surfactants,lubricants, etc. which are known to be used in the art. These can beselectively used according to the unit dosage form of the preparation.

As the unit dosage form of the pharmaceutical preparation, a variety offorms can be selectively used. The representative forms are tablets,pills, powders, solutions, suspensions, emulsions, granules, capsules,and suppositories.

The carrier which can be used in the processing into the tablet formincludes various excipients such as lactose, sucrose, sodium chloride,glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose,silicic acid, potassium phosphate, etc.; binders such as water, ethanol,propanol, simple syrup, glucose solution, starch solution, gelatinsolution, carboxymethylcellulose, hydroxypropylcellulose,methylcellulose, polyvinylpyrrolidone, etc.; disintegrators such ascarboxymethylcellulose sodium, carboxymethylcellulose calcium,low-substituted hydroxypropylcellulose, dry starch, sodium alginate,agar powder, laminaran powder, sodium hydrogencarbonate, calciumcarbonate, etc.; surfactants such as polyoxyethylene-sorbitan fatty acidesters, sodium lauryl sulfate, stearic acid monoglyceride, etc.;disintegration inhibitors such as sucrose, stearin, hydrogenated cacaobutter, hydrogenated oils, etc.; absorption promoters such as quaternaryammonium bases, sodium lauryl sulfate, etc.; humectants such asglycerol, starch, etc.; adsorbents such as starch, lactose, kaolin,bentonite, colloidal silica, etc.; and lubricants such as purified talc,stearates, boric acid powder, polyethylene glycol, and so forth.

Furthermore, where necessary, tablets may be prepared in the formshaving the conventional coatings such as sugar-coated tablets,gelatin-coated tablets, enteric-coated tablets, film-coated tablets,etc., or in the form of double-layered tablets or multi-layered tablets.

The carrier which can be used in the formation of pills includes variousexcipients such as glucose, lactose; starch, cacao butter, hydrogenatedvegetable oils, kaolin, talc, etc.; binders such as gum Arabic powder,gum tragacanth powder, gelatin, ethanol, etc.; and disintegrators suchas laminaran, agar, and so forth.

The carrier which can be used in the formation of suppositories includespolyethylene glycol, cacao butter, higher alcohols, higher alcoholesters, gelatin, and semi-synthetic glycerides etc. The encapsulatedproduct can be manufactured generally by blending the bacteria of theinvention with various kinds of pharmaceutical carriers such as thosementioned above and filling the mixture into hard capsule shells or softelastic capsule shells in the conventional manner.

Furthermore, where necessary, colorant, preservative, flavoring,corrigent, sweetener, and other drugs can be incorporated into thepharmaceutical product of the invention.

The quantity of the microorganism of the invention to be incorporated inthe preparation of the invention is not particularly restricted but canbe judiciously selected from a broad range. The generally recommendedproportion is about 10⁸-10¹⁰ cells/g of the pharmaceutical preparation.

The method of administration of the above pharmaceutical preparation isnot particularly restricted but can be established according to thepreparation forms, various patient factors such as age, sex, etc. andthe severity of illness. For example, the tablets, pills, solutions,suspensions, emulsions, granules, and capsules are administered orallyand the suppositories are administered rectally.

The dosage of said pharmaceutical preparation can be judiciouslyestablished according to the method of administration, the patient'sage, sex and other factors, and the severity of illness but ispreferably about 0.5-20 mg/day in terms of the microorganism of theinvention, i.e. active ingredient, per kg body weight. This preparationcan be administered in 1-4 divided doses a day.

On ingestion (administration) of the composition of the invention, themicroorganism in the composition finds its way alive into the lowerdigestive tract or settles there as part of the intestinal flora,whereby the expected efficacy is expressed. In this connection, theparticularly preferred preparation form is the enteric-coated tablet,with which the microorganism can be transported to the intestineswithout being attacked by gastric acid.

The equol-producing lactic acid bacteria-containing composition of theinvention as obtained in the above manner is useful for the symptomaticprophylaxis and treatment of malaise and/or postmenopausal osteoporosisand climacteric disturbances in middle-aged and elderly women. Suchprophylaxis and treatment can be accomplished by administering aneffective amount of said composition of the invention to the middle-agedor elderly woman for whom it is required or causing them to ingest thesame. The effective amount mentioned just above is not particularlyrestricted insofar as it is sufficient to prevent and control thevarious manifestations of osteoporosis and climacteric disturbancesaccompanying malaise and/or menopause in middle-aged and elderly women.As a rule of thumb, however, the dosage can be generally selected sothat the amount of equol excreted in the urine of the person who hastaken the composition of the invention reaches at least 5 μmole (about1.2 mg)/day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation showing the relationship ofincubation time to viable cell count as determined by the test protocoldescribed in Test Example 1.

FIG. 2 is a diagrammatic representation showing the relationship ofincubation time to equol-producing ability (score) as determined by thetest protocol described in Test Example 1.

FIG. 3 is a diagrammatic representation showing the relationship ofincubation time to equol output as determined by the test protocoldescribed in Test Example 1.

FIG. 4 is a diagrammatic representation showing the time course ofconcentration of each of daidzein compounds and equol in the culture asmonitored in accordance with the protocol described in Test Example 2.

FIG. 5 is a diagrammatic representation showing the relationship ofstorage period to equol-producing ability (score) as determined inaccordance with the test protocol described in Test Example 3.

FIG. 6 is a diagrammatic representation showing the incubationtime-dependent behaviors of the equol-producing strain (changes ingrowth performance, equol-producing ability, and equol output) asdetermined by the experiment described in Test Example 1-3.

FIG. 7 is a diagrammatic representation showing the relationship ofincubation time to viable cell count as determined by the test protocoldescribed in Test Example 4.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples of production of the equol-producing lactic acidbacteria-containing composition of the invention are intended todescribe the present invention in further detail and should by no meansbe construed as defining the invention.

EXAMPLE 1

(1) Production of a Fermented Soy Milk Beverage

The following ingredients were taken according to the formula andblended to prepare the composition of the invention in the form of afermented soy milk beverage.

Fermentation culture of 100 mL water-soluble soy protein Vitamins &minerals q.s. Flavoring q.s. Water q.s Total 150 mL

The above fermentative culture of water-soluble soy protein was obtainedby dissolving 13 g of water-soluble soy protein in 100 ml of water,adding 10⁸-10⁹ cells of Lactococcus 20-92 (FERM BP-10036), and carryingout fermentation at 37° C. for 24-48 hours. The water-soluble soyprotein used contained about 1-2 mg, calculated as daidzein, of daidzeincompounds in each one gram.

(2) Production of a Fermented Milk

The following ingredients were taken according to the formula andblended to prepare the composition of the invention in a fermented milkform.

Lactococcus 20-92 fermented milk 100 mL Vitamins & minerals q.s.Flavoring q.s. Water q.s. Total 150 mL

The Lactococcus 20-92 fermented milk was obtained by adding 10⁸-10⁹cells of Lactococcus 20-92 (FERM BP-10036) to 1 L of cow's milk (havinga nonfat milk solids content of 8.5% or greater and a milk fat contentof 3.8% or greater) and carrying out fermentation at 37° C. for 24-48hours.

(3) Production of a Freeze-dried Powder of Fermented Soy Milk

Using about 10⁹ cells of Lactococcus 20-92 (FERM BP-10036) and 100 g ofsoy milk (soy solids 10%, daidzein compound content 10-15 mg calculatedas daidzein), lactic acid fermentation was carried out at 37° C. for72-96 hours for the production of equol. This fermentation product wasfreeze-dried to prepare a powder. The equol content of the powder asdetermined by HPLC was 0.1-0.3 weight %.

The powder obtained above and various other ingredients were weighed outaccording to the following formula and blended to prepare thecomposition of the invention in the powder form (food form andpharmaceutical product form).

Freeze-dried powder of fermented soy milk 2.2 g (equol content 0.005 g)

Excipient (corn starch) 17 g Vitamins & minerals q.s. Flavoring q.s.Total 20 g(4) Production of a Powder

The following ingredients were weighed out according to the formula andblended to prepare the composition of the invention in a powder form(food form and pharmaceutical product form).

Freeze-dried powder of Lactococcus 20-92 4.1 g Excipient (lactose) 1.0 gVitamins & minerals q.s. Flavoring q.s. Total  20 g

A freeze-dried powder of Lactococcus 20-92 was obtained by culturingLactococcus 20-92 (FERM BP-10036) in a suitable liquid growth medium(MRS) (37° C., 24-48 hrs), harvesting and suspending grown cells in 10%skim milk, and lyophilizing the suspension. The cell content of thepowder was 10⁹-10¹⁰ cells/g.

The above powder may be made into a daidzein-containing powder byblending it further with 4.1 g of semi-purified soy isoflavine powder.

Intake of the daidzein-containing powder thus obtained results in theurinary equol excretions of about 5 μmoles (about 1.2 mg) per day,indicating clearly that the amount of equol corresponding to the aboveexcretions can be produced in vivo.

(5) Production of Granules

The following ingredients were weighed out according to the formula andblended to prepare the composition of the invention in a granular form(food form and pharmaceutical product form).

Semi-purified soy isoflavone powder 4.1 g Freeze-dried powder ofLactococcus 20-92 1.0 g Sucrose acid ester q.s. Vitamins & minerals q.s.Flavoring q.s. Total  20 g

The freeze-dried powder of Lactococcus 20-92 used was the same as theone used above in (1).

Intake of the above composition results in the concurrent delivery ofdaidzein and equol-producing bacteria to the large intestine, thusenabling production of equol in the large intestine.

Test Examples relating to the equol-producing lactic acid bacterialstrain of the invention are presented below.

TEST EXAMPLE 1

Test for Growth Performance, Equol-producing Ability (Activity), andEquol Output

(1) Test Protocol

Lactococcus 20-92 (10⁷-10⁹ cells/g) was incubated in 5 mL of BHI broth[a liquid medium for growth (basal medium)] anaerobically at 37° C. for24 hours and the culture was diluted to 10² and 10⁴ cells with the basalmedium.

The culture obtained at completion of incubation and its dilutionsprepared above were respectively taken, 0.2 mL each, and blended with 5mL each of daidzein-supplemented basal medium (daidzein added to BHIbroth at a final concentration of 10 μg/mL), cow's milk and soy milk,respectively, and cultured anaerobically at 37° C. The incubation timewas set to 8, 24, 48, 72, and 96 hours in the case of 10 μg/mLdaidzein-supplemented basal medium and soy milk, and 8, 24 and 48 hoursin the case of cow's milk.

Before the start of incubation and at the end of each incubation period,0.1 mL and 0.2 mL portions of the culture were sampled and respectivelysubjected to the counting of cells and assay of equol-producing ability(activity). Furthermore, for 10 μg/mL daidzein-containing basal mediumand soy milk, 0.5 mL of each culture was sampled before the start ofincubation and at the end of each incubation period and the amount ofequol produced in each sample was determined.

The number of bacteria was determined in the following manner. Each 0.1mL sample was diluted with PBS(−) (product of Nissui Co.) to prepare10⁴, 10⁵, 10⁶ and 10⁷-fold dilutions and 0.1 mL each of these dilutionswere respectively coated on GAM agar medium and incubated aerobically at37° C. for 24 hours. The colonies formed on the medium were counted foruse as the number of bacteria.

The equol-producing ability (activity) was assayed as follows. Each 0.2mL sample was blended with 5 mL of daidzein-supplemented basal medium(each in triplicate) and incubated anaerobically at 37° C. for 96 hours.At completion of incubation, 0.5 mL samples of the respective cultureswere taken and respectively extracted twice with 5 mL portions of ethylacetate and the daidzein, dihydrodaidzein (intermediate), and equol inthe extract were quantitated by HPLC. Moreover, based on the totalamount, the percentage of equol was calculated. The results were scoredon the following 5-point scale and the average score of 3 samples wasused as an index of equol-producing ability (activity).

4: Equol (90% or greater)

3: Equol produced, with daidzein diminishing to less than 50% (formationof intermediate)

2: Equol produced, residual daidzein (50% or greater) (formation ofintermediate)

1: Intermediate formed, equol not produced

0: Neither intermediate nor equol produced, with daidzein notdiminishing

The amount of equol produced was determined as follows. Each 0.5 mLsample was extracted twice with 5 mL portions of ethyl acetate and theamounts of daidzein, dihydrodaidzein (intermediate), and equol in theextract were quantitated by HPLC. Then, the respective concentrationswere used to calculate the amount of equol produced.

(2) Test Results

(2-1) The Results of Counting of the Cells (Growth Performance) arePresented in FIG. 1.

In the diagrammatic representation, (1) represents the result obtainedin the case where the daidzein-supplemented basal medium was used, (2)represents the result obtained in the case where soy milk was used, and(3) represents the result obtained when cow's milk was used. In eachdiagram, the horizontal axis represents incubation time (hr) and thevertical axis represents viable cell count (Log CFU/mL).

It can be seen from the respective diagrams, the growth performance ofthe strain of the invention is good and, regardless of the inoculum sizeused, the stationary phase of growth was invariably attained in 8 hoursof incubation in all the daidzein-supplemented basal medium, soy milkand cow's milk. The viable cell count was found to be steady at10^(9.1-9.4) CFU/mL in the daidzein-supplemented basal medium,10^(8.5-8.7) CFU/mL in soy milk, and 10^(8.0-8.4) CFU/mL in cow's milk.

(2-2) The Equol-producing Ability (Activity) Values Found are Presentedin FIG. 2.

In FIG. 2, (1) represents the result obtained in the case where thedaidzein-supplemented basal medium was used, (2) represents the resultobtained in the case where soy milk was used, and (3) represents theresult obtained in the case where cow's milk was used. In each diagram,the horizontal axis represents incubation time (hr) and the verticalaxis represents activity score.

It is obvious from the results presented in FIG. 2 that theequol-producing ability (activity) tends to increase with time in any ofthe daidzein-supplemented basal medium, soy milk, and cow's milk. Itcould also be confirmed that even in the cases where cow's milk and soymilk were used, the equol-producing ability (activity) of the strain ofthe invention is sustained.

(2-3) Results of the Amount of Equol Produced Determination

The quantities of equol produced in the daidzein-supplemented basalmedium and soy milk (about 80 μg/mL calculated as daidzein) were asshown in FIG. 3.

Referring to FIG. 3, (1) represents the result obtained in the casewhere the daidzein-supplemented basal medium was used and (2) representsthe result obtained in the case where soy milk was used. In eachdiagram, the horizontal axis represents incubation time (hr) and thevertical axis represents equol concentration (μg/mL).

In both media, the production of equol began to be noticed at hour-48following the start of incubation. In the case where soy milk was used,the amount of equol produced varied with inoculum size and particularlyat the inoculation level of 4.00%, the production of equol was as largeas 57.0 μg/mL at hour-96 of incubation.

Although, in soy milk, not less than 90% of daidzein serving as theprecursor of equol is present in the form of glycoside (in the form ofglucose attached), the peak corresponding to the glycoside was no longerobserved on the post-incubation chromatogram and this fact suggests thatthe strain of the invention decomposes the glycoside (β-glucosidaseactivity) to give daidzein and further metabolizes this daidzein toequol.

TEST EXAMPLE 2

Equol Production Pathway in Lactococcus 20-92

(1) Test Protocol

Lactococcus 20-92 (10⁷ cells/mL) was aerobically cultured in 5 mL of BHIbroth (a liquid medium for growth, basal medium) at 37° C. for 24 hoursand 0.2 mL of the resulting culture was blended with 5 mL ofdaidzein-supplemented basal medium and the mixture was incubatedanaerobically at 37° C. The incubation time was set to 8 hr, 24 hr, 30hr, 36 hr, 48 hr, 51 hr, 54 hr, 60 hr, 84 hr, and 96 hr.

Before the start of incubation and at the end of each incubation period,0.5 mL samples were taken and the concentrations of daidzein,dihydrodaidzein (intermediate), and equol in each sample weredetermined.

(2) Results

The results obtained are presented in FIG. 4.

FIG. 4 is a diagrammatic representation of the changes with time in theconcentrations of daidzein (left), dihydrodaidzein (center), and equol(right). In each diagram, the horizontal axis represents incubation time(hr) and the vertical axis represents the concentration (μg/mL) of thecorresponding substance.

It will be apparent from the data presented in FIG. 4 that theconcentration of daidzein began to decline at hour-48 of incubation, theintermediate compound dihydrodaidzein was formed during the period fromhour-48 to hour-60, and the production of equol began at hour-48. It canalso be confirmed that the metabolism of daidzein to equol hadsubstantially gone to completion by hour-60.

While the above results indicated that the metabolism from daidzein toequol occurred via said intermediate compound dihydrodaidzein, theresults also suggested that the formation of dihydrodaidzein and themetabolism thereof to equol took place in parallel.

TEST EXAMPLE 3

Low-Temperature Stability of Lactococcus 20-92 Strain-containingFermented Milks

(1) Test Protocol

Lactococcus 20-92 was cultured in 5 mL of a liquid medium for growth(basal medium) anaerobically at 37° C. for 24 hours, the resultingculture was used to inoculate 1 L and 2 L of cow's milk and 1 L ofcommercial skim milk (10% solids), respectively, at the level of 4% andcultured aerobically under stationary conditions at 37° C. for 48 hours.The cultures were stored at 4° C.

In the case of cow's milk, the equol-producing ability (activity) wasmonitored on a weekly basis following completion of culture through week4 of low-temperature storage at 4° C. Furthermore, two of the tubes werereserved and stored till day-42 and day-51, respectively, and theactivity was determined in each case.

In the case of skim milk, the activity was determined at completion ofculture and at week-1 and day-34 of low-temperature storage at 4° C.

The activity scores before storage and at the end of each storage periodwere generated by the above-described method comprising inoculating 5 mLof 10 μg/mL daidzein-supplemented basal medium at the level of 4% (0.2mL) in triplicate, culturing the microorganism anaerobically at 37° C.for 96 hours, and determining the concentrations of daidzein,dihydrodaidzein (intermediate), and equol for activity scoring.

(2) Results

The results are presented in FIG. 5. In FIG. 5, the horizontal axisrepresents storage period (days) and the vertical axis representsactivity score.

It is apparent from this diagrammatic representation of results that, asfar as cow' milk is concerned, the equol-producing ability (activity) issustained to week-4 of low-temperature storage at 4° C. after completionof culture in both cases of 1 L and 2 L. Moreover, in the case of 2 L ofcow's milk, the activity was found to be sustained to day-51, that wasthe last day of monitoring of the storage stability at 4° C. In the caseof 1 L of commercial skim milk, too, the equol-producing ability(activity) was apparently sustained to day-34, the last day ofmonitoring of the low-temperature storage stability at 4° C. aftercompletion of culture.

The foregoing results indicate that the fermented milk prepared by usingLactococcus 20-92 is capable of retaining the activity even underlow-temperature storage conditions and, therefore, is also suitable forfood distribution.

The relationship of the growth performance of Lactococcus 20-92 to itsequol-producing ability (activity) and the amount of equol produced asdeducible from the results obtained in the above Test Examples 1-3 canbe diagrammatically represented as shown in FIG. 6.

Thus, although cultural conditions varied with different culture media,the equol-producing ability (activity) can be maintained in both thegrowth phase and the stationary phase. On the other hand, with regard ofequol output, it appears that the enzyme begins to, be expressed oractivated to produce equol after a certain lag time in the stationaryphase.

TEST EXAMPLE 4

Gastric Juice Tolerance Test of the Fermented Milk Prepared by usingLactococcus 20-92

(1) Test Protocol

Lactococcus 20-92 was anaerobically cultured in 5 mL of a liquid mediumfor anaerobic growth (BHI broth, basal medium) at 37° C. for 24 hours.The resulting culture (10⁹ cells/g) was used to inoculate 1 L of cow'smilk at the 4% level and incubated aerobically under stationaryconditions at 37° C. for 48 hours. After completion of culture, the milkwas stored at 4° C. and, regarding it as fermented milk, was subjectedto the following test.

As artificial gastric juices, 0.045% pepsin-supplemented 50 mMglycine-HCl buffers (pH 2.5 and pH 3.0) were prepared. As control, 50 mMglycine-HCl buffer (pH 6.0) was prepared.

To 9 mL of each artificial gastric juice, 1 mL of the fermented milkstored at low temperature was added and the mixture was incubated(cultured) aerobically under stationary conditions in an incubator at37° C.

The incubation time was set to 1 hr, 2 hr, and 3 hr, and 0.1 mL and 0.2mL aliquots of each culture were respectively sampled before the startof incubation and at the end of each incubation period and subjected tothe determination of viable cell count (in the case of 0.1 mL) andequol-producing ability (activity) (in the case of 0.2 mL).

The determination of viable cell count was carried out in accordancewith the procedure described above in Test Example 1-(1), whichcomprises sampling 0.1 mL of each culture, diluting the sample 10⁴, 10⁵,10⁶ and 10⁷-fold with Nissui's PBS(−), coating 0.1 mL of each dilutionon GAM agar medium, incubating the inoculated medium aerobically at 37°C. for 24 hours, and counting the colonies formed on the GAM agar.

The determination of equol-producing ability (activity) was carried outin accordance with the procedure described above in Test Example 1-(1),which comprises inoculating 5 mL of daidzein-supplemented basal mediumwith 0.2 mL (4%) of the sample (in triplicate), incubating theinoculated medium anaerobically at 37° C. for 96 hours, and measuringthe concentrations of daidzein, dihydrodaidzein (intermediate), andequol in the medium for activity scoring.

(2) Results

The results obtained are presented in FIG. 7-A (viable cell count) and B(equol concentration).

In FIG. 7-A, the horizontal axis represents incubation time (hr) and thevertical axis represents viable cell count (Log CFU/ml in milk).

In FIG. 7-B, the horizontal axis represents incubation time (hr) and thevertical axis represents equol activity (score).

The following can be deduced from the results presented in FIGS. 7-A and7-B. Thus, in the buffer solution at pH 6.0, the viable cell count wassustained at the 10⁸ cells/mL level up to hour-3, and, here, theequol-producing ability (activity) was also sustained. In the artificialgastric juice at pH 3.0, the viable cell count was sustained up tohour-3 of incubation and, here, the activity was also sustained. On theother hand, in the artificial gastric juice at pH 2.5, a marked decreasein viable cell count began to take place at hour-2 of incubation and theactivity also disappeared.

When studied in the same test system as above, the probiotics(microorganisms which find their way alive into the intestinal canal andexhibit physiological activity there) on the market are reportedlyunchanged in viable cell count at pH 3.0 but decreased significantly atpH 2.5. This means that the tolerance to gastric juice at pH 3.0 allowsthese microorganisms to pass through the stomach alive. Therefore, thefermented milk prepared by using Lactococcus 20-92 is reasonablyexpected to deliver the organisms alive to the intestines to let themexhibit sustained activity in the lower part of the small intestine andin the large intestine.

TEST EXAMPLE 5

Bile Tolerance Test of Lactococcus 20-92

The tolerance to bile was determined with VITEK GPI Card (NipponBiomérieux Co., Ltd) and evaluated by using the growth performances ofthe strain of the invention in 10% and 40% biles as indicators.

(1) Test Protocol

Lactococcus 20-92 (10⁸⁻⁹ cells) was smeared on 5% sheepblood-supplemented Trypticase-Soy agar and aerobically cultured at 37°C. for 24 hours. The colonies on the medium at completion of culturewere hooked with a platinum loop and a homogeneous suspension thereof in0.5% sterile saline was prepared. This suspension was placed in VITEKGPI Card and, after 15 hours' incubation at 35° C., the growthperformance of the strain in the presence of bile was evaluated by usingthe dye (pH indicator). The bile was prepared by dissolving apredetermined amount of bile powder in sterile distilled water andplaced in the Card in advance.

(2) Results

The results of the above test indicate that Lactococcus 20-92 grows in10% and 40% biles, showing tolerance to 40% bile.

TEST EXAMPLE 6

Hemolysis Test of Lactococcus 20-92

(1) Test Protocol

Lactococcus 20-92 (10⁸⁻⁹ cells) was smeared on 5% sheepblood-supplemented Trypticase Soy agar and cultured anaerobically(N₂:CO₂:H₂=8:1:1) at 37° C. for 24-48 hours. The portion around thecolony formed on the medium after completion of culture was observed,and the hemolytic potential was evaluated according to the extent ofdecomposition of blood components (depigmentation or discoloration).

(2) Results

As the result of the above test, depigmentation (appearance of atransparent, colorless zone) was not observed around the colony,indicating that Lactococcus 20-92 does not cause β-hemolysis and, inthis respect, is a safe microorganism.

TEST EXAMPLE 7

Cell-infiltrating Enzyme Activity Test of Lactococcus 20-92

Regarding the systemic invasion of ingested lactic acid bacteria, adepression in the defensive function of the mesenterium or impairment ofthe mesenterium itself may be considered as the factor on the host side.As the factor on the side of bacteria, their enzymatic activity(cell-infiltrating enzymes) decomposing the lipid-protein complexproteoglycans constituting the mesenterium can be mentioned.

This test was intended to investigate whether Lactococcus 20-92 hascell-infiltrating enzyme activities, namely collagenase (gelatinase),hyaluronidase and sialidase (neuraminidase) activities, or not and wascarried out in the following manner.

(1) Test Protocol

Lactococcus 20-92 was smeared (smear size: 10⁸⁻⁹ cells) onblood-supplemented agar medium and cultured anaerobically(N₂:CO₂:H₂=8:1:1) at 37° C. for 24-48 hours.

The colonies on the medium after culture were hooked up with a platinumloop and suspended in sterile distilled water to prepare a homogeneoussuspension. Using this suspension, the presence or absence ofcollagenase (gelatinase) was investigated with Api™ (Nippon BiomérieuxCo., Ltd.) using the degradation of gelatin as the indicator.

Moreover, the test as to whether Lactococcus 20-92 has hyaluronidase andsialidase (neuraminidase) activities or not was carried out by theprocedure comprising incubating Lactococcus 20-92 in Tris-HCl buffersolution (pH 7.0) containing hyaluronic acid or sialic acid as thesubstrate (37° C., aerobic, 15 min. for sialidase activity and 24 hr forhyaluronidase activity) and measuring the degrees of decrease in theconcentrations of the respective substrates.

(2) Results

Lactococcus 20-92 showed none of collagenase (gelatinase),hyaluronidase, and sialidase (neuraminidase) activities.

Thus, in view of the fact that the strain of the invention lackscell-infiltrating enzymes which constitute a factor in infectivity, thestrain was confirmed to be a highly safe microorganism from infectivitypoints of view as well.

TEST EXAMPLE 8

Vancomycin Resistance Test

The acquisition of resistance to antibiotics (mutation) by bacteria hasbeen a matter of serious concern in recent years. It is not rare thatpatients infected with bacteria which have acquired such resistance toantibiotics succumb to death because they do not respond to theantibiotics. Particularly, the emergence of vancomycin-resistantbacteria (VRE) is a matter of serious concern in the field of clinicalmedicine today. Moreover, there is the apprehension that if the ingestedorganisms harboring the vancomycin resistance gene reach and settle inthe intestines where they come into contact with virulent or infectiousmicroorganisms (pathogenic bacteria), the vancomycin resistance gene maybe transferred to the pathogenic bacteria, with the result that thesebacteria also acquire vancomycin resistance. Therefore, it is necessary,at least, that microorganisms which are used as probiotics should not bevancomycin-resistant organisms.

This test was intended to investigate the susceptibility of Lactococcus20-92 to vancomycin and was performed as follows.

(1) Test Protocol

The vancomycin susceptibility test was performed using Sensi-Disk(product of Nippon Becton-Dickinson Company, Ltd.). Lactococcus 20-92was smeared (smear size: 10⁸⁻⁹ cells) on GAM agar medium, a diskcontaining 30 μg of vancomycin was placed on the medium, and an aerobicculture was carried out at 37° C. for 24 hours. After the aboveincubation time, the diameter of the inhibition zone formed around thedisk was measured and evaluated according to the evaluation table.

(2) Results

The diameter of the inhibition zone for Lactococcus 20-92 was 11.9±0.2mm and the susceptibility evaluation according to the evaluation tablewas positive (susceptible: =10 mm). This result indicated that thestrain of the invention is not a vancomycin-resistant strain and,therefore, is considered safe.

Presented in Example 2 below is an example of the production of equolfrom daidzein by using the lactic acid bacterial strain of theinvention.

EXAMPLE 2

Production of Equol

One mL of suspension containing 10⁷-10⁹ cells of Lactococcus 20-92 (FERMBP-10036) in GAM medium for culture of anerobic bacteria was preparedand this suspension was added to 100 g of soy milk (solidsconcentration: ca 2.2%). The mixture was incubated anaerobically at 37°C. for 72-96 hours and the equol produced in the culture was monitoredby HPLC. The daidzein compound content of said soy milk was 95 μg/mLcalculated as daidzein.

The result indicated the formation of 10.7±6.3 μg/mL (mean±standarddeviation of 3 experiments) of equol in the above soy milk culture.

The above finding shows clearly that by exploiting the microorganism ofthe invention, equol can be produced from the daidzein compoundscontained in food materials with good efficiency and at low cost.

The invention claimed is:
 1. An isolated strain of Lactococcus garvieae20-92 deposited under accession number FERM BP-10036, wherein saidstrain is in a lyophilized form.
 2. A composition comprising thelyophilized strain of claim
 1. 3. A composition comprising Lactococcusgarvieae 20-92 deposited under accession number FERM BP-10036, whereinsaid composition is in the form of fermented milk or fermented soy milk,said fermented milk or fermented soy milk having been fermented by saidLactococcus garvieae 20-92.